An important technique currently used in bioanalysis and in the emerging field of genomics is the polymerase chain reaction (PCR) amplification of DNA. As a result of this powerful tool, it is possible to start with otherwise undetectable amounts of DNA and create ample amounts of the material for subsequent analysis. PCR uses a repetitive series of steps to create copies of polynucleotide sequences located between two initiating (“primer”) sequences. Starting with a template, two primer sequences (usually about 15-30 nucleotides in length), PCR buffer, free deoxynucloside tri-phosphates (dNTPs), and thermostable DNA polymerase (commonly TAQ polymerase from Thermus aquaticus), these components are mixed, and heated to separate the double-stranded DNA. A subsequent cooling step allows the primers to anneal to complementary sequences on single-stranded DNA molecules containing the sequence to be amplified. Replication of the target sequence is accomplished by the DNA polymerase, which produces a strand of DNA that is complementary to the template. Repetition of this process doubles the number of copies of the sequence of interest, and multiple cycles increase the number of copies exponentially.
Since PCR requires repeated cycling between higher and lower temperatures, PCR devices must be fabricated from materials capable of withstanding such temperature changes. The materials must be mechanically and chemically stable at high temperatures, and capable of withstanding repeated temperature changes without mechanical degradation. Furthermore, the materials must be compatible with the PCR reaction itself, and not inhibit the polymerase or bind DNA.
Conventional PCR is typically carried out in tubes, microplates, and capillaries, all of which could be sealed conveniently. However, the geometry of these tubes, microplates, and capillaries render them not suitable for evanescent wave detection methods.
There are two common strategies to increase the signal to noise ratio when using evanescent wave detection methods. One method increases the real hybridization signal. The other method reduces the background signal.
There are a variety of technical solutions that can be employed order to increase the real hybridization signal. One known solution utilizes more sensitive fluorescent labels. Another known solution increases the hybridization efficiency by modifying exposure conditions like buffer compositions & temperature, or using a detector with high signal to noise ratio.
However, these technical solutions are not able to solve the problem completely or cause other problematic issues. As an example, using more sensitive fluorescent labels may also increase the background noise. In addition, altering exposure conditions may decrease the amplification efficiency and high quality detectors are generally cost prohibitive.
Some causes and solutions relating to high background signals in evanescent wave sensing have been discussed in several technical papers. As an example, M. Yoshida et al. 1993 Meas. Sci. Technol. 4 1077-1079 describe shifting to a longer excitation wavelength process in order to increase sensitivity an order of magnitude higher than that obtained in conventional system. In addition, facets of the substrate may be fine polished in order to decrease the scattering at the optical substrate surface. WO 2008/092291 A1 also describes how a multi-layer reflective or absorptive coating may be coated on the adhesion area on the bottom side of the substrate to prevent any scattering caused by an adhesive.
The above described solutions are only able to eliminate some of the unwanted fluorescent background signals that are generated within the reaction buffer where there is a high concentration of fluorescent molecules. The remaining unwanted fluorescent background signals typically come from four different aspects.
One aspect is the inherent noise of the detector. This aspect is extremely hard to clear up.
A second aspect comes from the interface between the cover plate and the reaction buffer. The interface causes non-specific binding between the fluorescent labeled DNA molecules and the cover plate surface. Attempts have been made to reduce non-specific binding by various surface modification methods which increase the inert characters of cover plate surface (e.g., by pre-hybridization). See Methods in Enzymology: DNA Microarrays, Volume 410, p 1.57, by Alan R. Kimmel, Brian Oliver.
A third aspect relates to inside the reaction buffer where there is scattering of excitation light such that excitation light travels into the reaction buffer in different directions. The scattered excitation light does not get totally reflected on the interface between the cover plate and reaction but instead causes excitation/emission of the fluorescence molecules inside the reaction buffer. WO 2008/092291 A1 describes modifying the cover plate by polishing or using a multi-layer reflective or absorptive coating to prevent the scattering.